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Cited by 130Tardigrade Biology and EcologyPhysiological and biochemical adaptationsPolar Research and Ecology

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Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

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Tanaka, Sae, Tanaka, Junko, Miwa, Yoshihiro, Horikawa, Daiki D., Katayama, Toshiaki, Arakawa, Kazuharu, Toyoda, Atsushi, Kubo, Takeo, Kunieda, Takekazu·2015·PLOS ONE
DOI 10.1371/journal.pone.0118272PMC PMC4326354
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Abstract

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

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Fig 1 Protein structure and mitochondria-targeting potential of RvLEAM. (a) Schematic representation of RvLEAM protein structure with hydropathy plot. Pink box indicates Mitochondria Targeting Peptide (MTP) at the N-terminus and the nine green boxes indicate LEA motifs found in RvLEAM protein. The six yellow bars in the hydropathy plot indicate predicted helix regions (H1–6) (b) Alignment of LEA motif sequences of RvLEAM with consensus. Greek characters in the consensus sequence indicate the following: Ψ, basic residue; Ω, acidic or amide residue; Φ, aliphatic residue; X, non-conserved residue. Residues consistent with the consensus are highlighted in deep green, and amino acids at non-conserved positions are shown in light green. Sequence logo created from nine LEA motifs of RvLEAM is shown below the alignment. (c) Amphipathic distribution of amino acids in predicted helical region of repeating LEA motifs 3 and 4. Amino acids are indicated as hydrophilic (pink), negatively-charged (red), positively-charged (blue), and hydrophobic (yellow) residues. (d) Subcellular localization of RvLEAM-GFP fusion protein (top) and GFP alone (bottom) in human HEp-2 cells. GFP signals (left). Mitochondria stained with Mito-Tracker (centre). Merged images (right).
Fig 1 Protein structure and mitochondria-targeting potential of RvLEAM. (a) Schematic representation of RvLEAM protein structure with hydropathy plot. Pink box indicates Mitochondria Targeting Peptide (MTP) at the N-terminus and the nine green boxes indicate LEA motifs found in RvLEAM protein. The six yellow bars in the hydropathy plot indicate predicted helix regions (H1–6) (b) Alignment of LEA motif sequences of RvLEAM with consensus. Greek characters in the consensus sequence indicate the following: Ψ, basic residue; Ω, acidic or amide residue; Φ, aliphatic residue; X, non-conserved residue. Residues consistent with the consensus are highlighted in deep green, and amino acids at non-conserved positions are shown in light green. Sequence logo created from nine LEA motifs of RvLEAM is shown below the alignment. (c) Amphipathic distribution of amino acids in predicted helical region of repeating LEA motifs 3 and 4. Amino acids are indicated as hydrophilic (pink), negatively-charged (red), positively-charged (blue), and hydrophobic (yellow) residues. (d) Subcellular localization of RvLEAM-GFP fusion protein (top) and GFP alone (bottom) in human HEp-2 cells. GFP signals (left). Mitochondria stained with Mito-Tracker (centre). Merged images (right).CC BY · PubMed Central
Fig 2 Mitochondrial localization of RvLEAM protein in tardigrade embryo. Tardigrade embryos were collected with eggshells and immunohistochemical analysis was performed using 10-μm frozen sections. Images show a representative section that was simultaneously reacted with anti-RvLEAM antiserum (a, d) and anti-ATP5A (mitochondrial ATP synthase) antibody (b, e). (c, f) Merged images. (d-f) Magnified images corresponding to the white boxes in (a-c).
Fig 2 Mitochondrial localization of RvLEAM protein in tardigrade embryo. Tardigrade embryos were collected with eggshells and immunohistochemical analysis was performed using 10-μm frozen sections. Images show a representative section that was simultaneously reacted with anti-RvLEAM antiserum (a, d) and anti-ATP5A (mitochondrial ATP synthase) antibody (b, e). (c, f) Merged images. (d-f) Magnified images corresponding to the white boxes in (a-c).CC BY · PubMed Central
Fig 3 Heat-soluble property of RvLEAM protein. Heat-solubility of RvLEAM protein is shown using either RvLEAM-induced bacterial lysate (a) or purified RvLEAM protein (b). ‘S’ indicates starting sample before heat treatment. After heat treatment, the proteins were separated into a soluble fraction (HS) and insoluble precipitate (HP). Arrows indicate RvLEAM protein.
Fig 3 Heat-soluble property of RvLEAM protein. Heat-solubility of RvLEAM protein is shown using either RvLEAM-induced bacterial lysate (a) or purified RvLEAM protein (b). ‘S’ indicates starting sample before heat treatment. After heat treatment, the proteins were separated into a soluble fraction (HS) and insoluble precipitate (HP). Arrows indicate RvLEAM protein.CC BY · PubMed Central
Fig 4 Subcellular localization of a novel mitochondrial heat-soluble MAHS protein. (a) Subcellular localization of MAHS-GFP fusion protein in human HEp-2 cells. (b) Heat-solubility of MAHS protein using bacterial lysate (left) or purified protein (right). Starting samples before heat treatment (S), soluble fraction after heat treatment (HS), and insoluble precipitate (HP) are shown. Red arrows indicate MAHS protein. (c) MAHS protein structure with hydrophathy plot. Mitochondria Targeting Peptide and conserved MAHS motif are indicated by the pink box and red box, respectively. Yellow and blue bars in the hydropathy plot indicate predicted helix regions (H1–5) and strand regions (S1–7), respectively. (d) Sequence alignment of conserved MAHS motifs among three tardigrade species, R. varieornatus, H. dujardini (Hd), and M. tardigradum (Mt). Pink and yellow indicate hydrophilic and hydrophobic residues, respectively (d) Amphipathic nature of predicted helix in MAHS motif. Amino acids are coloured as described in Fig. 1c.
Fig 4 Subcellular localization of a novel mitochondrial heat-soluble MAHS protein. (a) Subcellular localization of MAHS-GFP fusion protein in human HEp-2 cells. (b) Heat-solubility of MAHS protein using bacterial lysate (left) or purified protein (right). Starting samples before heat treatment (S), soluble fraction after heat treatment (HS), and insoluble precipitate (HP) are shown. Red arrows indicate MAHS protein. (c) MAHS protein structure with hydrophathy plot. Mitochondria Targeting Peptide and conserved MAHS motif are indicated by the pink box and red box, respectively. Yellow and blue bars in the hydropathy plot indicate predicted helix regions (H1–5) and strand regions (S1–7), respectively. (d) Sequence alignment of conserved MAHS motifs among three tardigrade species, R. varieornatus, H. dujardini (Hd), and M. tardigradum (Mt). Pink and yellow indicate hydrophilic and hydrophobic residues, respectively (d) Amphipathic nature of predicted helix in MAHS motif. Amino acids are coloured as described in Fig. 1c.CC BY · PubMed Central
Fig 5 Effects of RvLEAM and MAHS proteins on metabolic activity of human cells under hyperosmotic stress. Human HEp-2 cells (black), and HEp-2 cells expressing RvLEAM (light grey) or MAHS (dark grey) were exposed to medium supplemented with defined concentrations of sucrose for 48 h. After replacement of the medium with serum-free isotonic medium, cell metabolic activities were analysed using PrestoBlue reagents. Metabolic activities at each sucrose concentration are shown as a percentage of the mean activity observed in the cells at 0 mM sucrose (n = 3, ±SEM). Statistical significance against untransfected HEp-2 cells was determined by Dunnett’s test (*, P<0.05; **, P<0.01). P-values are shown above asterisks where statistically significant.
Fig 5 Effects of RvLEAM and MAHS proteins on metabolic activity of human cells under hyperosmotic stress. Human HEp-2 cells (black), and HEp-2 cells expressing RvLEAM (light grey) or MAHS (dark grey) were exposed to medium supplemented with defined concentrations of sucrose for 48 h. After replacement of the medium with serum-free isotonic medium, cell metabolic activities were analysed using PrestoBlue reagents. Metabolic activities at each sucrose concentration are shown as a percentage of the mean activity observed in the cells at 0 mM sucrose (n = 3, ±SEM). Statistical significance against untransfected HEp-2 cells was determined by Dunnett’s test (*, P<0.05; **, P<0.01). P-values are shown above asterisks where statistically significant.CC BY · PubMed Central

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Metadata

Authors
Tanaka, S., Tanaka, J., Miwa, Y., Horikawa, D. D., Katayama, T., Arakawa, K., Toyoda, A., Kubo, T., & Kunieda, T.
Affiliations
  • The University of Tokyo (JP)
  • University of Tsukuba (JP)
  • Keio University (JP)
  • Database Center for Life Science (JP)
  • National Institute of Genetics (JP)
Corresponding author
Kunieda, Takekazu
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-02-12
Volume
10
Issue
2
Pages
e0118272
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0118272
PMC
PMC4326354
OpenAlex
W1964745114
Cited by
128
References
46
Citations by year
2026: 7 · 2025: 8 · 2024: 32 · 2023: 15 · 2022: 13 · 2021: 16 · 2020: 7 · 2019: 12 · 2018: 6 · 2017: 7 · 2016: 4 · 2015: 1
FWCI
7.02
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Ministry of Education, Culture, Sports, Science and Technology (No. 221S0002, 221S0002)
  • Japan Society for the Promotion of Science (26660288, 25281016, 24657069, 13J10474, No. 221S0002, 221S0002)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Ecology, Evolution, Behavior and Systematics › Tardigrade Biology and Ecology
  • Physical Sciences › Environmental Science › Ecology › Physiological and biochemical adaptations
  • Physical Sciences › Environmental Science › Ecology › Polar Research and Ecology
Keywords
tardigrades, Ramazzottius varieornatus, anhydrobiosis, late embryogenesis abundant proteins, mitochondrial protection, desiccation tolerance, mitochondrial localization, human cells
MeSH terms
Amino Acid Sequence, Animals, Hot Temperature, Humans, Mitochondria, Molecular Sequence Data, Osmotic Pressure, Solubility, Amino Acid Motifs, Protein Transport, Mitochondrial Proteins, Tardigrada, Osmoregulation

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